The invention provides a method of introducing two or more genes of interest present on independent vectors into plant cells. The method of the invention employs an Agrobacterium-mediated gene delivery system by which multiple genes together with a single selectable marker gene are simultaneously transferred and inserted into the genome of plants with high frequencies. The ability to introduce foreign genes into plants is a prerequisite for engineering agronomic traits in plants. Many systems have been developed for introducing a foreign gene into plant cells, which involve mainly either Agrobacterium- or microprojectile bombardment-mediated transformation (Christou, 1996). The principle of all these systems involves the insertion of a target gene into the host plant genome together with a selectable marker gene encoding either antibiotic or herbicide resistance to aid in the selection of rare transgenic cells from non-transgenic cells. These systems are effective for introducing a single target gene into the host plant.
To alter agronomic traits, which generally are polygenic in nature, various genes involved in complex biosynthetic pathways must be introduced and expressed in plant cells. In this context, the traditional single-gene transfer systems are essentially useless for the following two reasons: 1) It is impractical to introduce multiple genes by repetitive insertion of single genes into transgenic plants due to the time and effort required for recovery of the transgenic tissues. In particular, a repetitive single-gene approach is highly impractical for plant species such as trees which, depending upon the species, require two to three years for transgenic tissue selection and regeneration into a tree; and 2) The presence of a selectable marker gene in a transgenic line precludes the use of the same marker gene in subsequent transformations of plant material from that line. Moreover, the number of available marker genes is limited, and many plant species are recalcitrant to regeneration unless appropriate antibiotic or herbicide selection is used.
Chen et al. (1998) recently reported the genetic transformation of rice with multiple genes by cobombardment of several gene constructs into embryogenic suspension tissues. However, particle bombardment-mediated gene transfer into embryogenic tissues is highly species-dependent, and regeneration of whole plants from embryogenic cells cannot be achieved for a variety of plant species (Horsch et al., 1985).
In contrast, Agrobacterium-mediated gene transfer and whole plant regeneration through organogenesis is a simple process and a less species-dependent system than bombardment-mediated transformation and regeneration via embryogenesis. However, the introduction of more than one gene in a single plasmid vector via Agrobacterium may be technically troublesome and limited by the number or the size of the target genes (Chen et al. 1998). For example, Tricoli et al. (1995) reported the transfer of three target genes to squash via Agrobacterium-mediated gene transfer. A binary plasmid vector containing the three target genes was incorporated into an Agrobacterium strain which was subsequently used to infect the leaf tissue of squash. As only one line was recovered from numerous infected squash tissues that contained all of the target genes, the use of a single binary vector with a number of genes appears to be a highly inefficient method to produce transgenic plants with multiple genes. Therefore, it is commonly accepted that transfer of multiple genes via Agrobacterium-mediated transformation is impractical (Ebinuma et al., 1997).
Thus, what is needed is an improved method to introduce multiple genes into plant cells.